I Ktioa hoc Nong nghiep
Xay dyng mo hinh ehuven gen
thong qua vi khuSn Agrobacterium liiniefaciens cho nSm dirtfc lieu Cordyceps militaris Gil
I r a n \ iSn Tuan' •'. \ u \ u a n Tao= ' Kh,.a Soih h... Inn-.c l\a h... K>^ j tt Tir 'Jucn. DsU / " " 0'"'' - ' " ' ^ "
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^- U K - . , -IJ j . - i o - ' 0 - n i i a v c h a p i i h a n d . - i i i g 2 9 ' 4 2 0 2 0 . ngav gin phan bivii 27 3 : o : i i . ngjv nh.;n phan bien - 4 4 - 0 - U . nLa> i
N'g.is nhyn bai 2
Tdm tdt:
Cordrceps militaris la mpt Inai n^n. diri^c li^u quv va da diryc chirng minh IA co chira nhif'u hoat ^,'*^^ co Igi cho su'c khoe. nhir adenosine, cordvcepin. p e n L a . i n . p»lvsaccharide. caroicnoid. Do do. loai n a m nay g a n day ^ ^ ^ c co. a n,6 hinh trien V^HR de nghien ciru ca ehC- p h a n tir cua qua trinh hinh thanh i,ua the va sinh tong h o p cac c h a t c6 h o a t tinh sinh hyc. p L . trien cAc hf- thCing ehuven s-cn hi^-u qua a C miliiaris se s o p p h a n q u a n t r g n g t r o n g viec t h u c h.?n cae UKhien eih. cai hii-n di Iruv^-n loai nAm dir^e li^u nav. T r o n s nghien eiru hien tai. mgt e h u n g C mdttans t h u t h ? p tai \ i(-t Nam dirg-c dinh d a n h c h m h xac v C- miit phan loai dtra tren eac dac diem hinh thai va gia. t n n h tir c u n g I T S cua rl)N \ . C h u n t ; miv din^e dSt (cn la C. militaris C12 v a dirge vu d u n s df nghien cuu chuyen gen. C h u n g C. tnilitaris (.12 dirg-e d a n h sia vC- mire dg mSn cam voi ha loai khang sinh pho bien d u n g trong chuyen gen la h y g r o m y e i n , nourseiUhriein va phlcomvcin. Kel qua eho tliiU chung C. militaris G12 chi hi uc che boi h y g r o m y c . n ff n o n g d o 200 f.g/ml. Ilivu qua ehuvC-n gen vao chung C. militaris Gil nhir vi khuan Agrobacterium tumefaciens vol m a r k e r chgn Igc lii -ien k h a n g h v g r o n n c i n d a l Irung binh 800 the chuvin gen/10' bao tir. Vice c h u y e n t h a n h c o n g gen chi thj DsRed V a GFP vao h^ gen cua C. militaris C; 12 da dirge \ a c nhan bSng PCR v6i cac c a p moi d a c hieu.
Tir khda: chu.v en gen thfing qua v t khuan .-Igrobacierium tumefaciens. Cordyceps miliiaris, m a r k e r k h a n g h y g r o m y e i n , vector n h | (hi*.
Chi so phdn loai: 4.6
Conivccps miliiaris la mOl loat nam kv sinh. dirge su dyng nhicu irong > hoc co iruvon nhir mot toai dugc lieu quy. t ' o hem 4110 loai Coiih,cps da dirge lim ihay va mo ta. Tu> nhien chi nioi so Inai nhai dinh dugc nuoi irong pho bien o dteu kicn nhan lao dc san vual sinh khoi qua ihe [ I - ,'] \."ini C tniliraiis da dugc chung minh la co kha nang khang khuan. khang n;im. khang ung ihu. khang viem. dieu hoa mien dich, chong o\v hoa [1-6]. Dieu do m a r a nhi^u tiem ning uiig dung ciio loat nam nav phuc v u cham soc sue khoe con nguai. Gtin da>. hat hgp chal co hoai linh khans ung ihir la cordvcepin va pcntosiaiin do mpl nhom cac gen chuyen bii;l chm irach nhicm sinh long hgp da dugc phai hien CO mat Irong he gen cua C. imliians [7]. Tuy nhicn.
niing lire stnh long hcrp cac chai co Igi cua cJc chuna lu nhicn ihirtmg lutmg Joi ihap \*iec can ihiep vao he sen
nam bang ky ihuiil di Iruyen co ihe giup tang hieu suat sinh long hgp cac chal co hoat tinh sinh hgc m o n g muon, Mgl Irong nh&ng phuong phap chuyen gen pho bien ho irg cho kv ihual chinh sua he gen a nam ia phirong phap chuyen thong qua vl khuan Agrobaclerium tumefaciens. Phirong phap nay da dugc chiing minh la rat hieu qua doi voi nhieu Ioai nam soi khac nhau [8]. Cho den nay, mgl so nghien ciru ve ehuven gen vao nam C. miliiaris sir dung vi khuan A.
tumefaciens da dugc thuc hien ihanh c o n j ,< Trung Qu6c Tuv nhien. hieu qua chuyen gen vao C uul,/, _ hua cao va con tiiy ihugc vao chiing nam d u g c su duni; 9. U)]. Trong nghien ciru nay, chiing nam C. mililaiis (.,\2 phan lap lai
\"ici Nam duoc xac nhan chinh xac vc nial ph.n, ir,ai de phuc v u c h o nghien ciru chuyen gen. Vice c h u j . , . r, vao chiing C militaris G12 da thanh cong voi phu.nvj • , , i^jy^^
gen nho vi khuan .4 tumefaciens su dun^ u. . _, |,<,n ]oc la gen khang khang sinh hygromyein.
, v-'.v/l.-'Wh- Ir:
S!S2I«rt\'s\am"<^'"°^° 60
Khoa hpc Ndng nghipp •
Establishment of an Agrobacterium tumefaciens-
mediated gene transfer approach for the medicinal fungus Cordyceps militaris G12
Van Tuan Tran'--', Xuan Tao \u--'
•Faculty of Biology-. UnivecityofScieiue.
Viutncim Notional Uiuvei sit-\. Hanoi Kev Laboraloiy ojEnzyme and Pmlein Technology.
University of Science. I'lelnani National Universitv. Hanoi 'Center of Experimental Biology, \aiinnal Center lor Tei. Imological
Progress. Ministry ol S( leiu e und Technology Received 2"; March 2(120: accepled 29 April 2020
Abstract:
Cordyceps militaris is a valuable edible and medicinal mushroom, which has been demonstrated to possess beneficial ingredients such as adenosine, eordycepin, piMitostatin, polysaccharides, and carotenoids.
Therefore, this fungus rcp.-esents a promising eell factory for investigating the .nolecular mechanism of fruiting body formation and biosynthesis of bioactive metabolites. The development of efficient transformation systems in C. militaris plays an important role in the genetic manipulation of this fungus. In the picscnt study, a commercial strain of C. militaris isolated in \'ietnam has been identified on the basis of morphological characteristics and the ITS (internal transcribed spacer) sequence of ribosomal DNA. This strain was designated as GI2 and employed for the genetic transformation.
Assavs for antifungal sensitivity of the C. militaris G12 sliain towards three antifungal compounds including hygromyein, nourseothricin. and phleonivcin revealed that this C militaris strain was inhibited only by hygromyein at the concentration of 200 fig/ml. Under optimised conditions for the genetic transformation of the strain G12 using the hygromyein resistance marker and .igrobacierium /n/He/oc/VH.v-mediated transformation method, the transformation efficiency could reach about 800 transformants per IO* spores. Further, the transfer of the DsRed Jii\6 GFP reporter genes into the genome of C. militaris G12 has been demonstrated to be successful hv PCR analyses with the gene-specific primers.
Kevword.s: .Agrobacterium tumefacien.s-mediated transformation, binarj vectors. Cordyceps militaris,, hygromyein resistance marker.
I 'at lieu nghien ciru
Mau qua ihc nam (kv' hicu G12) ihu ihap lir co sa nueii Irong nam C^ miliiaris 6 Ha Neii duoc dung dc phan lap chung nam lhuan khiel diing cho nghien ciru.
\'eclornhi [hepPK.2-Red [11] \apGrecn2 [12] mang cau true gen khang hygromyein va gcii chi thj DsRcd hoac GFP duoi sg dieu hoa ciia promoter gpd.4 lir nam .Aspeigillus nidulans dugc cung cap bm Phong Genomic. Phong Ihi nghiem Trgng diem Cong nghe Enzym va Protein. Truang Dai hgc Khoa hgc Tu nhien, Dai hgc Quoc gia Ha Ngi,
Cac cap moi su dung irong nghien ciru nay dugc licl kc chi liel irons; banu I,
Bang 1. Cac moi si> dung trong nghien CLFU.
Ton moi
ITSI ITS.}
GFP-F GFP-R IJsRe(!-F
IXsRcd-R
Trmhtv-(5'-3')
TCCGTAGGTGAACCTGCGG TCCTCrGCTTATTOATATGC- ATGCTGAGCAAGGCiCCiAG TCACrrGTACAGCTCGTC'CATGC A ACTCGAGCACGTGC1TAAGGAT ATCATGGCCTCCTCCGACiG AAGGATCCCCGCGGGAGCTC'GAT ATCCTACAGGAACAGGTGGTGGC
Kich IhirAe .un ph^m tbp) _ -550
72(1
739 113]
[ M l
1141
HPH-F HPH-R
CCGGTGACTCTITCTCiGC CTATTCCTTTGCCCTCGG
Classification number: 4.6
Phan lap vd quan sdt hinh thdi
Tach 1 phan sgi qua the nam, lam sach so bg b^ng nuoc cat va khir Iriing be mai qua Ihe bang con. Sau do phan sgi qua ihe dugc cai bo o hai dau, phan giCfa dugc cai ihanh cac lai nho va dal Icn moi Iruong PDA (polalosc dextrose agar) chira chloramphenicol (100 f^g/ml). Khang sinh chloramphenicol co tac dung irc che sinh truong ciia vi khuan. DTa dugc u 6 23"C trong loi 5 ngay. He sgi nam mau trang xual hien lir cac lai qua the dugc lhuan khiel bang phuong phap lach bao tu dcrn va cay chuyen sang dTa moi irucmg PD.A. moi. Chiing lhuan khiet dugc nuoi cay true licp tren tieu ban hien vi vo iriing co chua moi truong PDA dc quan sat hinh thai ciia he sai nam va cuong sinh bao lu duai kinh hien v i quang hgc [11].
Xde nhgn chung GI2 bdng gidi trinh lir viing ITS
Chung nam G12 dugc nuoi irong moi trutmg dich ihe
PDB (potato dexlrose broth) de thu he sgi diing cho lach
chiel DNA long so. Viec lach chiel DNA long so dugc Ihuc
hien theo quy trinh nhom nghien ciru da cong bo Iruoc dav
[14] D\.-\ long so duoc diing lam khuon de khuech dai
I Khoa hpc N6ng nghidp
vung ITS cua r l ) \ \ bSng PCR \ o i cap moi da nang ITSl ITS4 (bang 1). San pham PCR tinh sach duoc i:iji mnh lu ben cong iv Isl BASE i Sing.ip<'rci Trinh tu ITS dugc -^o sanh vol dir lieu trong Ngan hang gen quoc te (GenBank) su dung chuong innh BL.AST \ a phan iich v o eav phal sinh chung logi bang phan mem Mi ( i - \ "
Ddnh gid mirc dg mdn cdni khdng sinh cua chung GIJ Moi Iruemg PDA dugc bo sung khang Mnh hvgromvcin (50. 100. 150. 200 Mg mil. nourseothricin (50. 100. 200 va 300 pg/ml) hoac phlcomvcin (50. 100. 200. 300 figmD- Nho 10 |il djch bao tu nam ( I n hao tu ml) len moi truong V a dTa dugc ii 6 23"C trong 7 ngav Nong J o khang sinh uc cho hoan loan sg sinh iruong cua chung G I 2 ^c duoc su d\ing dc chuyen gen,
Chiiyi-n gen vd xde nhgn cdc the chiiyHn gen Qtiv irinh ehuven gen vao C. miltiaris G12 nho vi klui.in .( tunicUu icn\ dugc lien hanh iheo Zheng va cpng s\r (2011) [9] vai sg dicu chinh ve thoi gian done nuoi Ciiy la 2,5 ngav Cy the nhu sau: vector nhj the pPK2-Rcd (ho;'ic pClicen2) duge biC'n nap v;io chung vi khuan .-(
Itimefaeiens AGLl bSng xung dien sir dung he thong Bio- Rad Gene Pulse Xcell'""' Elcetroporation Syslem. Vi khuan A tumefaciens AGL\ mang vector pPK2-Reddugc sudung cho chuyen gen v ao C miliiaris G12. Dich v i khuan AGL1 dirge pha loang va nuoi cam img trong moi Iruong co bo sung aeeiosvniigonc (200 jaM) de dal mat dp quang (OD) khoang 0.6-0.8, \'i khuan sau do dugc Iron vtri bao lu nam (10" hao ur ml) ihcn lv le 1:1 v a dugc trai deu len mang giay liH- cellulose phu san Iren dTa moi Iruong cam img. DTa duac li irong 101 a 23'C irong 2.5 ngav de ki'ch hoat qua trinh ehuven gen lir.-l, luinefaciens ^ang chung C. mUiiaris GI2 Mang gi.iv lge sau do duoc elniven sang moi irucmg PDA CO ho sung khang sinh hvgromycin (200 fig/ml) de chgn Igc cae khuan lac nam chuyen gen va bo sung khang sinh celblaxin-ic (300 jig/mi) de diet vi khuan A. tumefaciens Dia duge u o nhiet do 23-25 "C irong 7 ngay.
Cac the chuyen gen sau do dugc thuan khiel va DNA long so dugc lach ehiet phuc vu cho \ a c nhan the chuydn gen bang PCR voi cap m6i dac hieu HPH-F/HPH-R va DsRcd-FDsRed-R hoac GFP-F/GFP-R (bang 1).
K^t qua va thao luan
Chiing G12 dirffc xac nhdn thugc lodi C. mUitaris Tir mau qua the (hinh 1 .A). chung G12 da dugc phan lap 6 dang lhuan khiel. Sau 5 ngav nuoi eav tren moi iruong PD.'X. chiing n.ini smh truong lao he sgi miiu irang. Quan sal duoi kinh hien vi. he sgi bao gom eau iruc phan dot boi cac vach ngan (hmh I B . C. D). San 7 ngay nuoi c a \ . chum: G I 2 hinh ihanh bao lu don^. -.runy ihinh IE). Dua
ircn mau qua the mJu vang Jicn iiub thai quan sal duoc eo the- k h a r ^ dinli Coniyccps [\>. 16]. Dc Jam MO L).
G12 dimg cho nghien cuu ehuven get - l a i t r i n l u u vimg ITS cu.i r n N - \ ^ • i a : s S r R N \ eua i H N ^ I''' /^^
rRN.A: 1TS2) o nam co muc .!o ' loai eo quan he gan giii '•'"^' "''•' dugc su dung phii hicn o 'n^^
"f ^ ""^ ""^ :"::X, "Z ^^^^^ '^^-^ *•>• n^^' «> «•='•
„ c ™ . ASns 100% vm cac trinh .i, ITS cua C „„l„ans .0 .roncTco .^iiil lieu GenBanli. Phan lich mo phong cay phat smli^chung loai voi phin mem MEGA7 xac nhan chung Ihuoc loai C minimis (hinh IQ).
, , j . w J K m liinh , t^-,i: liuioc chi
; ; , h \ a c eua chting htm. KII nen hanh .i^. ^en ! 8 S r R N A
^•,,^{;,ITS1:5,8S ,0 hon giira cac
• J irinh tir nay i., ,. K-huech ._ I lir D N A tong
L Con/yaB' mtHirts soar HBRC 30
nvirsna laoulD VNS IKVOaiaSS 1)
Hinh 1, Phan lap va dinh danh chung C militaris G12. (A) Qua the n&m; (B, C) Chiing G12 Iren rr- - . pQ^ ^ ^ gj Cau true he sgi va bao iLf, (F, G) Phan tier "hung ioai Chung G12 dga tren Irinh tu' ITS ctia rDNA
Chung C. militaris G12 mdn edm vd liygromycin
De lua chon dirgc loai khang sinh cCinj khang sinh ihich hgp su dung trong sang Ig^
gen. chung C miliraris G12 d u g c nuoi ca% ^ CO bo sung khang sinh hygromyein, nou'
lihdng sinh
iv.ii ' i t i n g d o :^'- he chuyen
•^ '^oi-irirng
KHOAHOC CONG NGHE
Khoa hoc Nong nghi$p i
phlcomvcin vai cac nong do khac nhau. Ca ba khang sinh nay deu la nhirng khang sinh dugc sii dung pho bien trong nghien ciru chuyen gen 6 nam [8. 9, 11. 12. 18. 19]. Sau "
ngav nuoi a nhiet do 25°C. chiing C. miliiaris G12 khang voi nourseothricin va phleomycin 6 cac nong do tucmg doi cao la 50. 100, 200 va 300 pg/ml. Tuy nhien. sg sinh truong cua chung G12 bi irc che hoan loan boi hygromyein o nong dg 200 jig/ml (hinh 2). Cac nghien ciru gan day cho Ihay nhieu chiing thugc loai C miliiaris khang vcri khang sinh hygromyein, nhung mpi so chiing lai man cam \di hygromyein [9, 10], Trong nghien cuu nay. chiing toi chi ra rang chiing C miliiaris G12 man cam voi hygromyein cr nong dp 200 pg/ml va khang sinh nay phii hgp de sir dung cho cac thi nghiem chuyen gen vao chung G12.
he gen cua ca 6 chiing voi sir .\ual hien ciia cac hiing I l \ \ CO kich thuoc tuong ung (hinh 3B). \ h u vav, chimg toi da ihanh cong trong viec ehuven gen vao chiing C. militaris G12 su dung v i khuan 4. tumefaciens voi marker chgn Ipc la gen khang hygromyein. Hieu qua chuyen gen irong nghien cuu nay dat irung binh 800 the chuyen gen cho 10" bao lu va cao hem 1.3 lan so vol cong bo ciia Zheng va egng su (2011) [9]. Kel qua dal dugc cao hem nghien cuu truoc dav CO the la do thai gian dong nuoi cay trong quy trinh chuyen gen nav keo dai hem 0.5 ngay va chung G12 co IS cho hieu qua chuyen gen lot hon Mgt uu dicm niia ciia chting G12 la n6ngdgh\gromycin sir dung cho ehuyen gen thap hon 2 lan so vtji chung C miliiaris JM4 trong nghien ciru cita Zheng va cpng su(2011).
Hinh 2. Kha nang man cam khang sinh cua chting C. militaris GM.
Chuyen gen vdo chung C. militaris G12 sir dung vi khudn A. tumefaciens
De ihir nghiem chuyen gen vao chung G12 nhd .A liiniefaciens, vector nhi the pPK2-Red duge sir dung. Sau quy trinh chuyen gen, chiing toi da Ihu dugc rat nhieu khuan lac xuat hien tren moi Irucmg chpn Igc PDA co bo sung khang sinh hygromyein (hinh 3A). Siiu khuan lac ngau nhien (ky hieu CM-Rl den CM-R6) dugc chon de lhuan kliiel, xac nhan lai kha nang khang hygromyein. DNA long so ciia 6 chiing dugc tach chiet de dting cho PCR xac nhan su chuyen gen thanh cong. Hai cap moi HPH-R/HPH-F dac hieu cho gen khdng hygromyein va DsRed-F/DsRed-R dac hieu cho gen chi Ihi DsRed (bang 1) dutTc su dung cho PCR. Kel qua nuoi cay tren moi truong chpn loc xac nhan ca 6 chung ehuven gen deu khang vi^i hygromyein (hinh -vA). Them vao do. ket qua PCR ciing chi ra rang gen khang hygromyein la HPH va gen chi thi DsRed tir v i khuan A.
tumefaciens da duge chuyen va lich hgp thanh cong vao
Hinh 3. Xac nhan the chuyen gen DsRed. (A) Kel qua chuyen gen DsRed vao chiing C miliiaris G12 sic dung vi khu^n A.
tumefaciens. (B) Xac nhan chuyen gen thanh cong bSng PCR voi cap mbi HPH-R/HPH-F va DsRed-F/DsRed-R, trong do (-) la d6i Chung am. {+) la doi chirng du'ong.
Ket qua luong tu eQng nhan dugc khi thuc hien chuyen gen GFP vao chiing C militaris G12 sir dung vector nhi the pGreen2 (hinh 4A). Cac chimg (ky hieu CM-Gl den CM-G6) dugc xac nhan chuyen gen ihanh cong bang PCR su dung cap moi HPH-F/HPH-R va GFP-F/GFP-R, Ket qua chi ra rang, ca 6 chiing chuyen gen dugc lua chgn ngau nhien deu xuat hien 2 bang DNA tuong irng voi gen khang hygromvein HPH va gen chi thi GFP (hinh 4B}. \ h u vay.
ket qua da khang dinh viec chuyen gen thanh eong vao chung C. miliiaris G12 khi sir dung gen khang khang sinh hygromvein. Mo hinh chuyen gen Ihiel lap eho chung C mUtlaris G12 co nguon goc Viet Nam se !a co so quan trpng ho tro cho cac nghien cuu tiep Iheo nham kich hoat kha nang sinh long hgp eac hoat chat co loi a loai nam dugc lieu quan trgng nay.
63
I Khoa hpc Nong nghi^
Hinh 4, Xac nh§n th^ chuydn gen GFP. (A) Kel qua chuyen gen GFP v^o chiing C militans G12 sir dyng vi khuan A tumefaciens, (B) Xac nhgn chuyen gen thanh c6ng bSng PCR vt/'\ cap m6i HPH-F/HPH-R vA GFP-F/GFP-R
KAtlufin
Chiing G12 phan lap igi \ lei \ a i i i dugc xac nhan chinh
\.ic thugc loai Coniyceps miliiaris dira tren dac diem hinh lliai \ a giai trinh tg v ung ITS eua rON \ , Chting C. miliiaris G12 bj irc che hoan loan biji khang sinh hygromyein o nong dg 200 fig ml va khong man cam v6i khang sinh nourseothricin va phleomycin. \ g h i c n cim nav da chung minh v ii;c chuyen gen thanh eong vao ehung G 12 co nguon goc \ ' i c ' ""^^nii nhij VI khuan A. lunh-Uuiens vai marker chpn Igc la gen khang hvgromvcin Hieu qua chuyen gen vao chiing C militaris G12 dat trung binh 800 the chuyen gen • 10" bao lu,
l d i C A M 0N
Nghien ciru nay dugc ho Irg kinh phi boi Dai hoc Quoc gia Ha N'in thiing qua de tai ma so O G . 2 0 . i 9 "Nghien cuu phat trien cong nghe moi de lai tao. phuc hoi va duy tri cac chung nam dugc lieu Cordyceps militaris co hieu sual cao V e hinh ihanh qua the v a sinh long hop eordycepin" Cac lac gia xin Iran trgng cam on,
TAl Lieu THAM KHAO
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